Project Summary: This application details a five year research career development program designed to explore biofilm formation by the food-borne pathogen Listeria monocytogenes under the guidance of Dr. Roberto Kolter, Professor in the Department of Microbiology and Molecular Genetics at Harvard Medical School. Dr. Kolter is a leader in the field of microbial biofilm development and has an excellent track record of mentoring his trainees to positions as independent investigators. My long-term goal is to become an independent clinician-researcher. To this end, I have trained in both pediatric infectious diseases and biology. This proposal is designed to prepare me to be an independent researcher studying biofilm formation by L. monocytogenes. This field of study is very different from my prior research and my plan includes coursework in bacterial metabolism and bioinformatics. The gram-positive bacterium L. monocytogenes is a food-borne pathogen that causes severe, even fatal, infections in fetuses, newborns, and immunocompromised people. L. monocytogenes persisting on food preparation surfaces in the form of biofilms is a source of contamination that leads to listeriosis outbreaks. Biofilms are highly organized, matrixenclosed assemblages of microbial cells. Of particular relevance to both medicine and the food industry, biofilm bacteria are more resistant to detergents, biocides, and antibiotics than are individual, free-living bacteria. This makes decontamination of biofilm-coated surfaces particularly difficult. Although little is known about how Listeria forms biofilms, Listeria pathogenesis is well studied. As a result, a number of genetic and genomic tools are available. The experiments proposed herein are designed to determine: (1) what L. monocytogenes genes are required for biofilm formation using transposon and targeted mutagenesis, and (2) what are the physiological differences between individual free-living bacteria and biofilm-associated bacteria for L. monocytogenes using DNA microarray technology. Insights gained from these experiments should lead to new approaches to decontaminate biofilm-coated surfaces, and a better understanding of biofilms in general. Relevance: Prevention by eliminating sources of infection remains a cornerstone in the continued effort to decrease the human and economic toll of infectious diseases. Ingestion of food contaminated with Listeria monocytogenes can cause severe, sometimes fatal, infections in newborns, pregnant women and people with compromised immune systems. The research I have proposed will lay the foundation for understanding how Listeria monocytogenes persists in the environment in biofilms on surfaces, such as those in food processing centers, where it can be a source of infection. [unreadable] [unreadable] [unreadable]